Cell lysates and tissue homogenates were prepared using RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS) supplemented with 1X Protease Complete (Roche 11697498001) and Phosphatase Inhibitor Mini Tablets (ThermoFisher A32957). Proteins (20 μg) were separated by 4–12% Bis-Tris polyacrylamide gel electrophoresis and transferred to PVDF membranes using an iBLOT 2 system (ThermoFisher). Blots were blocked in 5% non-fat milk for 1 h at room temperature and probed overnight with antibodies against phospho-Akt (Cell Signaling 4060, 1:1000 dilution), Akt (Cell Signaling 4691, 1:1000 dilution), mTOR (Cell Signaling 2983, 1:1000 dilution), phospho-S6K (Cell Signaling 9234, 1:1000 dilution), S6K (Cell Signaling 2708, 1:1000 dilution), and GAPDH (Fisher AB2302MI; 1:10,000 dilution). Appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) were used to visualize proteins in combination with HRP substrate (Jackson Immunoresearch 703-035-155 and 111-035-144, 1:10,000 dilution).
Protease complete
Manufactured by Roche
Protease Complete is a comprehensive solution that provides a broad spectrum of proteolytic activity. It is designed to facilitate the efficient digestion and solubilization of a wide range of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions)
Iblot2 system, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Lithium hydroxide, by Merck Group (1 mentions)
Potassium persulfate, by Merck Group (1 mentions)
2 acrylamido 2 methylpropane sulfonic acid, by Merck Group (1 mentions)
Thermo ltq mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline pbs, by Corning (1 mentions)
Mueller hinton broth (mhb), by BD (1 mentions)
N isopropylacrylamide, by Merck Group (1 mentions)
Acrylic acid, by Merck Group (1 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
N n methylenebisacrylamide, by Merck Group (1 mentions)
Methyl acrylate, by Merck Group (1 mentions)
Ab38971, by Abcam (1 mentions)
A2547, by Merck Group (1 mentions)
Phosphostop, by Roche (1 mentions)
Spi1 pu 1, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Gapdh, by Biosynth (1 mentions)
7 protocols using protease complete
1
Western Blotting of Signaling Proteins
2
Liver and Cell Lysate Preparation
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To make liver lysates, liver tissue was disrupted with a Tissue Lyser (Qiagen) and steel beads in the presence of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Igepal (v/v), 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) plus 1X protease inhibitors (Protease Complete, Roche) at 4 °C. Cell lysates were made by resuspending cell pellets in RIPA buffer (approximately five pellet volumes) with protease inhibitors (0.5 μM phenylmethylsulfonyl fluoride, 5 μg/ml Pepstatin A, 10 μg/ml Leupeptin) on ice. Lysates were cleared by spinning in a microfuge at 21,130g for 10 to 15 min at 4 °C, and the supernatant was processed as the sample.
3
Preparation of Enriched Liver Membrane Fractions
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Liver membrane fractions were prepared as described previously (52 (link)) with some minor modifications. Frozen tissue (∼1 g) was thawed on ice in five tissue volumes of Homogenization Buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl, 100 mM EDTA) and homogenized with an immersion blender (Bamix) until homogenization was completed based on visual inspection of the sample. The homogenate was cleared by spinning at 10,000g for 30 min at 4 °C. The supernatant was spun at 100,000g for 90 min at 4 °C. The pellet was washed with Resuspension Buffer (100 mM sodium pyrophosphate, pH 7.4, 1 mM EDTA) and spun at 100,000g for 60 min at 4 °C. The pellet was defined as the enriched membrane fraction. It was resuspended in Storage Buffer (50 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 20% (v/v) glycerol) with 0.1 mM DTT to 10 to 20 mg protein/ml. DTT was omitted for samples intended for coimmunoprecipitation. All buffers contained protease inhibitors (Protease Complete, Roche) at 1X or at 2X for samples intended for coimmunoprecipitation.
4
Synthesis and Characterization of Antimicrobial Peptides
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The peptides used in these studies were custom synthesized by ChinaPeptides Company (Shanghai, China) and had purities of ≥ 95%, based on chromatographic analysis of the purified peptides. Synthetic peptides were verified on a Thermo LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The broad-spectrum protease inhibitor cocktail Protease cOmplete was purchased from Roche Diagnostic, Corp. (Indianapolis, IN). Resazurin, sodium salt is purchased from Sigma-Aldrich (St. Louis, MO). N-Isopropylacrylamide (NIPAm), N, N′-Methylenebisacrylamide (BIS), Acrylic acid (AAc), 2-Acrylamido-2-methylpropane sulfonic acid (AMPS), Methyl Acrylate (MA), Lithium hydroxide (LiOH) and potassium persulfate (KPS) are all purchased from Sigma-Aldrich (St. Louis, MO). Mueller Hinton Broth (MHB) was purchased from Becton Dickinson and Company (Sparks, MD). Phosphate buffered saline (PBS) was purchased from Corning-cellgro (Manassas, VA). Commercial alligator plasma was purchased from Bioreclamation (Westburg, NY). Alligator blood was acquired from St. Augustine’s Alligator Farm (St. Augustine, FL). All experiments involving alligators were carried out with compliance with relevant guidelines, using protocols approved by the GMU IACUC.
5
Protein Production Analysis of Primary Cell Isolates
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Protein production from primary cell isolates were assessed by immunoblot analysis as previously described [10 (link)]. Protein production was evaluated in cells from P2 or P3. Briefly, ~3 X 105 cells were seeded in tissue culture dishes in growth media and conditioned media and cell layers were collected at day 2 and 3 (and some cases 4 when allowable cell numbers were available) after plating. Cell layers were washed one time in PBS, collected in 1% deoxycholate with protease inhibitors (protease complete, Roche), and centrifuged at 10,000 x g. Proteins soluble in deoxycholate were separated by SDS-PAGE analysis and subjected to immunoblot analysis. Primary antibodies used were rabbit polyclonal anti-C-terminal propeptide of collagen I (LF-41, a kind gift of Dr. Larry Fisher, NIH) [11 (link)], rabbit polyclonal anti-telopeptide of collagen I (generated and verified at MUSC), rabbit polyclonal MTI-MMP (Chemicon, #AB8221 & Abcam, #ab38971), and mouse monoclonal against alpha smooth muscle actin (Sigma, #A2547). Antibodies against tubulin were used to establish equal loading of protein and to serve as a readout of baseline protein production for each culture. Primary antibodies were detected with appropriate secondary antibodies conjugated to horse radish peroxidase incubated with ECL reagent. Detection was captured using either X-ray film or a GE image documentation center.
6
Protein Extraction and Western Blotting
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Protein from whole cells was extracted using lysis buffer containing 0.3M sodium chloride, 1% (v/v) Triton X-100, and 50mM Tris pH 8.0 supplemented with ProteaseComplete (Roche) protease and PhosphoSTOP (Roche) phosphatase inhibitors. Western blotting was performed as described in the Supplementary Methods and supplementary table 4 (available online).
7
Western Blot Analysis of SPI1/PU.1
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Tissue was homogenized in RIPA buffer; phosphatase (Sigma) and protease (Complete®, Roche) inhibitors were added. Lysates were resolved by SDS–PAGE and immunoblotted for SPI1/PU.1 (Cell Signaling Technology, Leiden, the Netherlands) overnight at 4°C. GAPDH (Fitzgerald Industries, Huissen, the Netherlands) was used as a loading control. Imaging was performed on a ChemiDoc XRS+ System (BioRad, Temse, Belgium). Quantification was done using Image J software (NIH).
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